Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), is the leading cause of bacterial infectious disease mortality. The cell envelope of M. tuberculosis provides a permeability barrier, which shields the bacterium from environmental stress, provides intrinsic resistance to chemotherapeutic agents, plays a key role in TB-persistence, and participates in mycobacterial pathogenesis. The distinguishing feature of the mycobacterial cell wall is the presence of an outer waxy coat comprised of an extraordinary variety of exotic lipids. While lipid biosynthesis is essential, lipid catabolism also plays a key role in mycobacterial physiology and latency. M. tuberculosis encodes for an astonishing 34 fatty acid adenylating enzymes FadD's involved in lipid metabolism. By contrast E. coli encodes a single fadD. The extraordinary functional redundancy of the fadD's is truly amazing and clearly illustrates the importance of lipid metabolism. Based on sequence alignment and functional characterization, the mycobacterial FadD's have been divided into 2 classes of adenylating enzymes: 1) long chain fatty acyl-AMP ligases (FAALs) involved in lipid biosynthesis and 2) acyl-CoA synthetases (ACSs) involved in lipid catabolism. The identification of specific small molecule inhibitors against each class of FadD or selective inhibitors of an individual FadD is expected to help decipher the functional role of the FadD's play in lipid metabolism and could additionally lead to the development of new class of antitubercular agents. Toward this goal we propose to develop a fluorescence polarization assay amenable to high throughput screening to identify small molecule FadD inhibitors. Our long-term goal is to understand the functional role of the 34 fadD's encoded in the M. tuberculosis genome and how these contribute to mycobacterial pathogenesis. The objective of this application is to develop and validate a fluorescence polarization assays amenable to high-throughput screening of FadD32 (fadD32 is an essential gene required for mycolic acid biosynthesis and a representative member of the FAAL class of FadDs) and FadD19 (fadD19 is a gene implicated in cholesterol catabolism that is required for mycobacterial persistence and a representative member of the acyl-CoA synthetases class of FadD's). Compounds identified in the HTS assay will be validated by an established pyrophosphate exchange radio assay as a secondary screen. Subsequent counter screening against a panel of FadD's will access the selectivity of identified compounds, which may represent useful tools to interrogate mycobacterial lipid metabolism. PUBLIC HEALTH RELEVANCE: Mycobacterium tuberculosis the causative agent of Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. Additionally, M. tuberculosis and other atypical mycobacteria are now classified as opportunistic infections of AIDS patients. The proposed research is expected to enable identification of new antitubercular agents with novel mechanisms of action.